Multidrug resistance-associated protein 2 (MRP2) and oxaliplatin transport

Abstract

The platinum-based drug oxaliplatin is currently used in the clinical treatment of gastrointestinal cancer. Efflux membrane transporters have been reported to play roles in determining the cellular accumulation and activity of platinum-based drugs, but there have been few studies of oxaliplatin. We hypothesized that the efflux transporter multidrug resistance protein-2 (MRP2), encoded for by the ABCC2 gene, may play a role in determining cellular sensitivity to oxaliplatin by transporting oxaliplatin-derived platinum. Human MRP2-expressing inside-out membrane vesicles prepared from Sf9 insect cells were used for studies of oxaliplatin transport. A HEK293 cell line stably transfected to over-express the human ABCC2 gene (HEK-MRP2 cells) and its isogenic HEK293 parenteral (HEK-P) cell line, and a panel of seven human gastrointestinal cancer cell lines, were used to study the role of MRP2 in determining the cellular accumulation and activity of oxaliplatin, as assessed by inductively coupled plasma mass spectrometry and MTT growth inhibition assays. Membrane vesicle studies showed that MRP2 mediated the ATP-dependent active membrane transport of oxaliplatin-derived platinum [Vmax = 2680 pmol per mg of protein per 10 min (95%CI of 2010 to 3360 pmol per mg of protein per 10 min); Km = 301 μM (95%CI of 163 to 438 μM)]. Studies of oxaliplatin stability in membrane vesicle incubation buffer (oxaliplatin stability half-life = 2.24 hrs with 95% CI of 2.08 to 2.43 hrs) suggested that intact oxaliplatin and its early degradation product, [Pt(DACH)oxCl]-, were likely substrates for MRP2-mediated active transport. HEK293 cell line studies showed that cellular accumulation of oxaliplatin-derived platinum and sensitivity to oxaliplatin-induced growth inhibition were both reduced in HEK-MRP2 cells by up to two-fold compared to HEK-P cells, but these deficits were reversed by inhibition of MRP2 with myricetin. Studies of human gastrointestinal cancer cell lines demonstrated that MRP2-mediated deficits in cellular accumulation of oxaliplatin-derived platinum were observed in HepG2 and PANC-1 cells (0.14- and 0.34-fold difference relative to HEK-P cells), which had high expression levels of ABCC2 mRNA comparable to the HEK-MRP2 cells (1360- and 139-fold difference in relative to HEK-P cells). Myricetin increased cellular platinum accumulation of HepG2 and PANC-1 by 5- and 2.8-fold, respectively, and their sensitivity to oxaliplatin-induced growth inhibition by 3.4- and 4-fold, respectively. In HepG2 cells, siRNA-mediated knockdown of ABCC2 expression increased cellular accumulation of oxaliplatin-derived platinum by approximately 3-fold compared to control-siRNA treated HepG2 cells. Inhibiting MRP2 sensitised MRP2-overexpressing gastrointestinal cancer cells to growth inhibition induced by clinically achievable concentrations of oxaliplatin (3.75 to 11.25 μM). In conclusion, MRP2-mediates the active transport of oxaliplatin-derived platinum. Furthermore, MRP2 was identified as a targetable factor, limiting the cellular accumulation and anticancer activity of oxaliplatin in human gastrointestinal cancer.